Experimental Insights on the Use of Secukinumab and Magnolol in Acute Respiratory Diseases in Mice

This study investigates the combined treatment of secukinumab (SECU) and magnolol (MAGN) in a mouse model of LPS-induced ALI overlapped with allergic pulmonary inflammation, aiming to better understand the mechanism behind this pathology and to assess the therapeutic potential of this novel approach in addressing the severity of ALI. The combined treatment reveals intricate immunomodulatory effects. Both treatments inhibit IL-17 and promote M2 macrophage polarization, which enhances anti-inflammatory cytokine production such as IL-4, IL-5, IL-10, and IL-13, crucial for lung repair and inflammation resolution. However, the combination treatment exacerbates allergic responses and increases OVA-specific IgE, potentially worsening ALI outcomes. MAGN pretreatment alone demonstrates higher potency in reducing neutrophils and enhancing IFN-γ, suggesting its potential in mitigating severe asthma symptoms and modulating immune responses. The study highlights the need for careful consideration in therapeutic applications due to the combination treatment’s inability to reduce IL-6 and its potential to exacerbate allergic inflammation. Elevated IL-6 levels correlate with worsened oxygenation and increased mortality in ALI patients, underscoring its critical role in disease severity. These findings offer valuable insights for the advancement of precision medicine within the realm of respiratory illnesses, emphasizing the importance of tailored therapeutic strategies.


Introduction
Up to 4% of all hospital admissions and more than 10% of all intensive care unit (ICU) admissions are acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS) patients [1], with a mortality rate of up to 40-70% despite the improvements in intensive care [2], and with evidence of adversely affected long-term quality of life for patients who survive the acute phase of ALI [1].ALI is characterized by death or dysfunction of alveolar epithelial cells and/or pulmonary capillary endothelial cells, which leads to disruption of the alveolar-capillary barrier [3], while ARDS represents the clinical manifestation of diffuse, severe ALI with increased pulmonary vascular permeability, massive inflammation, and subsequent pulmonary edema and refractory hypoxia [4,5].Other authors consider ALI as a moderate or mild form of ARDS on a progressive scale [6].Moreover, there are indirect causative relations between asthma and ALI/ARDS which may also have practical implications on the clinical circumstances in view of the fact that pneumonia, along with aspiration pneumonitis and sepsis, is among the leading causes of ARDS [7].
Early stages of ALI in humans are characterized by elevated interleukin-17A (IL-17A) in circulation and in BALF, and neutrophils have an important role in recruitment, alveolar permeability, and organ dysfunction in ARDS [8,9].The Th17/Treg imbalance favoring a Th17 shift represents a potential risk indicator in patients with early ARDS due to its association with more pronounced organ dysfunction and worse oxygenation [10].In a mouse model of LPS-induced ALI, IL-17 aggravated lung inflammation and neutrophil infiltration [11].On the other hand, inhibition of IL-17 signaling pharmacologically or through genetical modification showed protective effects against development of experimental lung injury [12].
Magnolol (MAGN), a bioactive ingredient of Magnolia officinalis-Figure 1-is known for anti-inflammatory activity and for the ability to ameliorate endotoxin-induced multiple tissue damage and dysfunction [26,27], including the attenuation of pro-inflammatory cytokines in rodent models of LPS-induced ALI [28][29][30][31].Among the numerous studies concerning ALI/ARDS treatment, MAGN pretreatment has been noted for its ability to significantly improve severe lung damage (caused by lung edema, alveolar wall thickening, and neutrophil infiltration), to decrease the number of total leukocytes in BALF, and to diminish lung myeloperoxidase (MPO) activity in the lungs of rodents with LPS-induced ALI [28][29][30].

Introduction
Up to 4% of all hospital admissions and more than 10% of all intensive care unit (ICU) admissions are acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS) patients [1], with a mortality rate of up to 40-70% despite the improvements in intensive care [2], and with evidence of adversely affected long-term quality of life for patients who survive the acute phase of ALI [1].ALI is characterized by death or dysfunction of alveolar epithelial cells and/or pulmonary capillary endothelial cells, which leads to disruption of the alveolar-capillary barrier [3], while ARDS represents the clinical manifestation of diffuse, severe ALI with increased pulmonary vascular permeability, massive inflammation, and subsequent pulmonary edema and refractory hypoxia [4,5].Other authors consider ALI as a moderate or mild form of ARDS on a progressive scale [6].Moreover, there are indirect causative relations between asthma and ALI/ARDS which may also have practical implications on the clinical circumstances in view of the fact that pneumonia, along with aspiration pneumonitis and sepsis, is among the leading causes of ARDS [7].
Early stages of ALI in humans are characterized by elevated interleukin-17A (IL-17A) in circulation and in BALF, and neutrophils have an important role in recruitment, alveolar permeability, and organ dysfunction in ARDS [8,9].The Th17/Treg imbalance favoring a Th17 shift represents a potential risk indicator in patients with early ARDS due to its association with more pronounced organ dysfunction and worse oxygenation [10].In a mouse model of LPS-induced ALI, IL-17 aggravated lung inflammation and neutrophil infiltration [11].On the other hand, inhibition of IL-17 signaling pharmacologically or through genetical modification showed protective effects against development of experimental lung injury [12].
Magnolol (MAGN), a bioactive ingredient of Magnolia officinalis-Figure 1-is known for anti-inflammatory activity and for the ability to ameliorate endotoxin-induced multiple tissue damage and dysfunction [26,27], including the attenuation of pro-inflammatory cytokines in rodent models of LPS-induced ALI [28][29][30][31].Among the numerous studies concerning ALI/ARDS treatment, MAGN pretreatment has been noted for its ability to significantly improve severe lung damage (caused by lung edema, alveolar wall thickening, and neutrophil infiltration), to decrease the number of total leukocytes in BALF, and to diminish lung myeloperoxidase (MPO) activity in the lungs of rodents with LPS-induced ALI [28][29][30].Endotoxins or LPS are derived from the cell wall of gram-negative bacteria and are able to induce a sepsis syndrome associated with key features of ALI: lung recruitment of inflammatory cells, increases in capillary permeability, and subsequent alveolar edema [32].Previous authors have shown that experimental asthma with intratracheal OVA challenge of earlier OVA-sensitized mice notably exacerbated lipopolysaccharides (LPS)-induced infiltration of leukocytes in bronchoalveolar lavage fluid (BALF) [33].
The individual capacity of both MAGN and SECU in mitigating LPS-induced ALI has been extensively recorded; however, the underlying mechanisms and the specific types of immune responses involved have been only partially clarified.Furthermore, there exists a research gap in terms of a comparative analysis of these two molecules and their interaction, and considering their respective positive impacts on ALI, it can be presumed that a synergistic effect would result from their combination.
In the current investigation, we aimed to bring more clarity in this direction by using a particular murine model of LPS-induced ALI overlapped on OVA-induced allergic pulmonary inflammation, as this type of experimental ALI showed an increased level of severity [33].We evaluated the levels of cytokines specific to different types of immune responses, such as Th1, Th2, and Th17, following treatment with SECU and MAGN administered either alone or in combination.
As the effects of SECU treatment alone in comparison to saline vehicle control and dexamethasone treatment were previously investigated [18], the current paper is focusing only on the comparative evaluation of SECU treatment, MAGN treatment, and their association.

Preparation Protocol: 2% CMC-Na Mucilage and Magnolol Suspension
Two grams of CMC-Na powder were added in small quantities under stirring in 80 g of water heated to 65 • C, and stirring continued for 15 min until powder was completely dispersed in a water bath mortar.Up to 100 g was made with distilled water, homogenizing gently, avoiding air incorporation.Subsequently, it was allowed to stand for 24 h at room temperature for a complete hydration and structuring process, after 100 g of 2% CMC-Na translucent mucilage was obtained.
The MAGN suspension pharmaceutical formulation was made by dispersing magnolol 0.5% (5 mg/g) in diluted CMC-Na 1% mucilage (obtained by dilution with distilled water).The active substance was gradually dispersed in a small amount of 1% CMC-Na mucilage in the mortar using a pestle, and then 0.25% Tween ® 80 was added, stirred, and finally the last portion of mucilage was added by gentle homogenization for 5 min.The suspension for oral use was packaged in sterile plastomer containers and stored in the refrigerator for up to 10 days.

Animal Experimental Design
The Research Ethics Committee of the "Grigore T. Popa

Sensitization Protocol
Mice underwent a period of acclimatization to the internal environment for seven days before the onset of allergic asthma.The OVA grade V was solubilized to a final concentration of 20 µg/mL in 500 µL of sterile PBS per mouse, with the addition of aluminum hydroxide (alum) to a concentration of 2 mg/mL, following the protocol outlined by Debeuf et al. [35].The resulting mixture was subjected to rotation for 30 min at room temperature using an end-over-end rotator.The mice belonging to the OVA + LPS, OVA + LPS + MAGN, OVA + LPS + SECU, and OVA + LPS + MAGN + SECU experimental groups were intraperitoneally injected with the OVA/alum solution at a total volume of 0.2 mL per mouse on days 0 and 7, as indicated by prior research [35].Subsequently, on days 14, 15, 16, and 17, the animals underwent a 25 min challenge in an inhalation chamber with OVA by aerosolizing 10 mL of 1% OVA diluted in PBS for the OVA + LPS, OVA + LPS + MAGN, OVA + LPS + SECU, and OVA + LPS + MAGN + SECU groups, following the methodology outlined by Debeuf et al. [35] (Figure 2).

ALI Induction and SECU Treatment
On days 15 and 18, the intratracheal LPS administration was performed 1 h after OVA aerosol under general anesthesia with ketamine [80 mg/kg mouse bodyweight (BW)] and xylazine (10 mg/kg BW) intraperitoneally, as described in the protocol of Ehrentraut et al. [36].On day 18, all animals were euthanized in deep anesthesia (ketamine and xylazine overdose) by atlanto-occipital dislocation.

Drug Administration
MAGN at 50 mg/kg body weight was administered to the OVA + LPS + MAGN and OVA + LPS + MAGN + SECU groups daily via oral gavage, from the first day of OVA exposure (day 0) to day 18, as described by Huang et al. [37].On the days of OVA challenge or SECU treatment, mice received MAGN 60 min prior to each aerosol challenge or SECU injection, respectively.
On days 14, 15, 16, and 17, SECU was administrated at a dose of 10 mg/kg subcutaneously 1 h prior to each OVA aerosol [17].The doses of SECU were established from similar experiments on mice [17,38].

Sample Collection of Blood for OVA-Specific IgE
Mice were exsanguinated from the vena inguinalis immediately after loss of pedal withdrawal reflex (i.e., no response to a toe pinch).The blood was collected in 0.5 mL microcentrifuge tubes and the serum separated from whole blood by centrifugation (10 min; 3000 revolutions per min; 4 • C) for determination of OVA-specific IgE using a Legend Max Mouse OVA Specific IgE ELISA kit (Biolegend, Sandiego, CA, USA), an Infinite 200 PRO M Plex Tecan plate reader (Tecan, Grodig, Austria), and Magellan v 7.4 software (Tecan, Grodig, Austria).

Sample Collection of BALF for Cytokines
BALF was obtained via tracheal cannulation using three consecutive infusions/aspirations of ice-cold PBS (0.5 mL each), totaling 1.5 mL, followed by centrifugation at 250× g for 5 min at 4 • C. The resultant supernatant was then transferred to a new Eppendorf tube, centrifuged at 10,000× g for 15 min, divided into 150 µL aliquots, and preserved at −80 • C until cytokine assessment (RD-LXSAMSM-08 Luminex Mouse Discovery Assay 8-Plex: IFN-γ, IL-4, IL-5, IL-6, IL-13, IL-17/IL17A, TNF-alpha, VEGF).Following vortex mixing, 5 µL from the remaining cell pellet was utilized for slide preparation for differential counting, and subsequently stained with May-Grünwald (Sigma-Aldrich, 1.01424) and Giemsa (Sigma-Aldrich, 1.09204).The average cell count per microscopic field was calculated using an optical microscope with a dry objective lens (400× total magnification) by examining 10 fields.The identification of neutrophils, eosinophils, lymphocytes, and macrophages was carried out using an optical microscope with an immersion objective lens (1000× total magnification) by analyzing 200 cells.

Analysis of Cytokines in Lung Homogenate
The right pulmonary lobes were excised subsequent to the retrieval of bronchoalveolar lavage fluid, assessed for mass, and subsequently immersed in 200 µL of NP-40 lysis buffer (Thermo Fisher Scientific, UK) supplemented with protease inhibitor cocktail (Promega, USA) and placed on ice for a duration of 15 min.Subsequent to that, manual grinding was carried out on ice utilizing a 1 mL Wheaton Tenbroeck tissue grinder.The resulting homogenized mixture underwent centrifugation at 10,000× g for 15 min, followed by preservation of 60 µL portions of the resulting supernatant at −80 • C until further analysis.

Histological Analysis of Lung Tissue
Following the collection of BALF, the lower lobe of the left lung was excised and then placed in a solution containing 4% formaldehyde for fixation.The tissue was subsequently dehydrated using varying concentrations of ethyl alcohol, followed by clarification with xylene, impregnation, and embedding in paraffin.Sections of the tissue, measuring at 5 µm, were then prepared and stained using Hematoxylin Eosin (H.E.) as well as periodically with Schiff acid (PAS) for further analysis.The histological structures were examined and measured using a Leica microscope, and photomicrographs were taken for documentation and analysis purposes.

Statistical Analysis
The data obtained are presented as the means ± standard error (SE).Figures are depicted in bar formats along with standard error and asterisks denoting significant differences in comparison to the SAL group.In order to assess the statistically significant distinctions among multiple groups, the parametric one-way analysis of variance (ANOVA) was employed, followed by either the Holm-Šidák method for multiple comparisons or the non-parametric Kruskal-Wallis one-way analysis of variance on ranks.All statistical analyses were conducted using the Statistical Package for the Social Sciences (SPSS) version 27.0 (SPSS Inc., Chicago, IL, USA) software.A p-value of less than 0.05 was deemed to be statistically significant.In Figure 4, the alterations in inflammatory cells in BALF for macrophages, neutro-  In Figure 4, the alterations in inflammatory cells in BALF for macrophages, neutrophils, lymphocytes, and eosinophils are illustrated for each group.The depiction of the eosinophil in picture (f) exhibits remarkable similarities in aspect to the specific subtype of SiglecF + Gr1 hi eosinophils [39].Percentual quantification in BALF of differential cell counts for neutrophils, lymphocytes, macrophages, and eosinophiles reveals a significant decrease of neutrophils in the case of MAGN treatment solely (p < 0.05).On the other hand, MAGN showed the potential to slow down the decline of macrophages comparative to SECU treatment alone or to the positive disease control group (p < 0.05).The tendency of SECU to decrease macrophages can also be observed in the MAGN and SECU combined treatment (Figure 5).There were no statistically significant differences among the counts of lymphocytes and eosinophils in any of the groups.

Cytokines in BALF
Representative cytokines for specific Th1, Th2, and Th17 immune responses were evaluated in BALF for each investigated group (Figure 6).SECU treatment alone has clearly shown the ability to decrease the level of IL-6 in BALF.The association of MAGN pretreatment to SECU was not able to potentiate this effect on IL-6, but surprisingly did the opposite in the case of Il-13: SECU treatment paired with MAGN pretreatment had an inhibitory role on IL-13 increase in comparison to administration solely of MAGN that undoubtedly induced an increment of IL-13 in BALF.
Both SECU and MAGN, whether used alone or in combination, resulted in a reduction of IL-17.Unfortunately, the distinctions in their impacts do not reach statistical significance, thus making it challenging to determine comparatively whether any of them or their combination exerts a superior impact on IL-17.
Interestingly, while SECU treatment decisively decreases the level of INF-γ, MAGN pretreatment acts as a strong stimulus for IFN-γ increase.Even when it is administrated in addition to SECU, the stimulatory influence of MAGN categorically counteracts the inhibitory effect of SECU on IFN-γ.
In addition to the previously cited cytokines, the quantity of VEGF was assessed in BALF for each group.There was no notable alteration observed as a result of any of the treatments studied or their amalgamation (Table 1).To acquire a comprehensive understanding of the interaction among various immune reactions, the identical cytokines evaluated in BALF were measured in LTH (Figure 7).
VEGF evaluation in LTH revealed an increase under MAGN pretreatment compared to the positive disease control group.Unfortunately, the significance of this rise cannot be thoroughly and comparatively appraised due to statistically questionable data concerning the impact of SECU therapy alone on VEGF dynamics.
MAGN has demonstrated a highly efficacious stimulatory impact on IL-4 in this particular pathological induced setting, as both in monotherapy and combined with SECU therapy it led to a clearly discernible elevation of this cytokine.
Regarding the influence on IL-5, only the increase under combined treatment can be considered, as this is the only one measured as statistically noteworthy.However, the substantial rise appears to be predominantly attributed to MAGN.
Since IL-13 level is significantly raised in both the OVA + LPS + MAGN and OVA + LPS + MAGN + SECU groups in comparison to the positive disease control group, the enhancing impact can appropriately be ascribed to MAGN.
The impact of MAGN on the concentration of IFN-γ is undeniably characterized by a stimulating effect when compared to SECU.The robustness and potency of this influence are further underscored and substantiated by the fact that it continues to manifest itself even in the case of SECU co-administration.

Histological Analysis
The lung histological alterations induced by the presence of OVA + LPS involve an augmentation in the size and quantity of goblet cells in the pseudostratified ciliated epithelium that lines the airways.This epithelial layer exhibits disruptions in the connections between its cells, which are discernible under light microscopy.Additionally, there is edema around blood vessels and bronchial tubes, along with an infiltration of neutrophilic polymorphonuclear cells, eosinophils, lymphocytes, and peribronchial macrophages.Within the bronchial lumen, polymorphonuclear cells are visible.Alveoli in the lungs are collapsed, with an increase in the thickness of the septal walls.Neutrophils, monocytes, eosinophils, alveolar macrophages, and lymphocytes can be identified within both the septal walls and the alveolar lumens (Figure 8 and Table 1).In the context of SECU treatment only, a notable reduction is observed in the quantity of neutrophils, persisting at diminished levels in the vicinity of the bronchi.The pulmonary alveoli exhibit a diminished thickness in their walls and a decreased presence of figurative elements within the lumen or septal structures, and instances of alveolar collapse are infrequent.The OVA + LPS + MAGN group demonstrates a compact, reduced region in contrast to the OVA + LPS + SECU group, within the central zone of the lobules, where the pulmonary alveoli are compressed and predominantly invaded by neutrophils, lymphocytes, and macrophages.
The aspect of the OVA + LPS + SECU + MAGN group unveils even less pronounced pathological modifications compared to the OVA + LPS + MAGN group.
There is a consistent decrease of neutrophils, leading to a significant reduction in their presence, particularly around the bronchi.Furthermore, an observable characteristic of the lung alveoli within this group is the presence of a thinner wall, along with an obviously diminished count of figurative elements found either in the lumen or the septal wall.Additionally, the collapsing of the lung alveoli is a rare occurrence in this group.
The level of lung inflammation and caliciform cell hyperplasia was assessed subjectively on a scale ranging from 0 to 4: 75%.In order to quantify caliciform cells in bronchi and bronchioles, a five-point classification system was utilized, with categories including 0: <0.5% PAS-positive cells and 1: <25%, 2: 25-50%, 3: 50-75%, and 4: >75%.Each slide was evaluated by counting five fields, and the mean score was derived from observations of five animals.The quantification of PAS-positive caliciform cells was determined as the number of positive PAS cells per millimeter of basement membrane to adjust for the size of the airway [40].To mark inflammatory cell infiltration in the intraluminal, alveolar, peribronchial, and perivascular regions, cell counting was performed blindly based on a five-point grading system for the following characteristics: 0: normal, 1: few cells, 2: a ring of inflammatory cells, a deep cell layer; 3: a ring of inflammatory cells 2-4 cells deep, 4: a ring of inflammatory cells >4 cells deep [40].

Discussion
The current research assesses the immunomodulatory impacts of combined treatment with SECU and MAGN on some of the specific cytokines associated with the Th1, Th2, and Th17 axes of the immune response in a murine experimental model of LPS-induced ALI overlaid on OVA-induced allergic pulmonary inflammation.We choose this experimental model of particular degree of severity based on the apparent deleterious influence of preexisting allergic asthma on ALI outcome [33].The results of SECU treatment alone in comparison to the SAL (saline) and OVA + LPS + DEXA group (control group-positive for treatment) were previously published [18].
The increased OVA-specific IgE serum concentration under MAGN and SECU combined treatment, when contrasted with the levels in all the other groups exposed to OVA, appears to have a detrimental effect within the framework of ALI.
The effects of SECU and MAGN combined use on ALI at the level of lung tissue histology might be interpreted as a "moment picture" of the dynamic interplay among different branches of the immune system (Th1, Th2 or Th17), rather than as a definitive end outcome.
In the specific design of our ALI experimental model, the Th2 immune response is significantly overshadowed by the strength of the non-Th2 immune response triggered by the high-exposure dose of LPS.This immune response shift can be partly explained by the ability of IFN-γ to inhibit the baseline state of Th2 activation in subjects with asthma by mechanisms involving the inhibitory effect of type 1 cytokines on Th2 inflammation [41].
This predominance of non-Th2 response is contrasted with the increased levels of IgE.One possible reasoning could be the marked increase in IL-13, which, independently of IL-4, stimulates the maturation of B cells and significantly promotes IgE synthesis [42,43].
The tissular damage observed in the untreated group results primarily from the infiltration of neutrophils induced by the Th17 response.However, the mitigation of ALI effects at the level of lung tissue histology under combined use of SECU with MAGN has two possible components: IL-17 blockade by SECU and inhibitory effects of MAGN on neutrophils activity [44,45] MAGN pretreatment showed higher potency on neutrophil decrease than SECU in our experimental model of increased degree of severity of ALI given the preexistence of asthma.Neutrophils play a pivotal role in acute lung inflammation, primarily through their involvement in the pathogenesis of conditions such as ALI and acute respiratory distress syndrome (ARDS).Our results may indicate a favorable influence of MAGN pretreatment in relation to the degree of asthma severity.The utilization of MAGN pretreatment might ameliorate the symptoms experienced by individuals suffering from asthma.Other authors found that pretreatment i.p. with MAGN (5, 10 and 20 mg/kg i.p.) significantly decreased the number of neutrophils (p < 0.01) in BALF [29] and that SECU exerted beneficial effects in ALI inflammation [20,21].Upon activation, neutrophils migrate to the lungs, where they release a variety of pro-inflammatory mediators, including reactive oxygen species (ROS), cytokines, and proteolytic enzymes, which contribute to tissue damage and inflammation.Neutrophils also form neutrophil extracellular traps (NETs), which, while essential for trapping pathogens, contribute to ALI pathogenesis by releasing pro-inflammatory and cytotoxic molecules that exacerbate thromboinflammation and lung tissue injury [46].Additionally, IL-17 has been shown to enhance neutrophil functions, including ROS production and NET formation, thereby amplifying lung tissue damage during inflammation [47].Despite their critical role in host defense, the dysregulated and prolonged presence of neutrophils in the lungs can lead to severe tissue damage and impaired resolution of inflammation, highlighting the need for therapeutic strategies that can modulate neutrophil activity without compromising their protective functions.
Our study results revealed the presence of SiglecF + Gr1 hi eosinophils in BALF of the MAGN-treated group in optical microscopy (Figure 4-picture (f)).These eosinophils represent a distinct subpopulation within the lungs of allergen-challenged mice, characterized by a unique cytokine profile that includes lymphocyte-targeting cytokines, suggesting their pivotal role in modulating immune responses during allergic inflammation [39].These eosinophils express IL-13 at significant levels (72 pg/10 6 cells) [39], indicating their potential role in enhancing transition from a pro-inflammatory to an anti-inflammatory macrophage phenotype.Alternative macrophage activation is induced by type 2 cytokines, IL-4 and IL-13 [48,49].In the early stages of ARDS, classically activated (M1) macrophages dominate, secreting pro-inflammatory cytokines to clear pathogens, which may inadvertently damage alveolar epithelial cells and contribute to cell death.As the condition progresses, the presence of alternatively activated macrophages (M2s) becomes more pronounced.These M2 macrophages secrete anti-inflammatory cytokines that dampen the inflammatory response, thereby promoting epithelial regeneration and alveolar structure remodeling, crucial for the recovery phase of ARDS [50].Their ability to switch from a pro-inflammatory phenotype in the early stages of injury to a pro-repair phenotype is crucial for effective lung repair [51].The metabolic activity of M2, influenced by immunometabolism, plays a significant role in their function during lung repair.The metabolic intermediates produced by M2s support their polarization and function, highlighting the importance of their differentiation in the regulation of macrophage activity during lung repair [49].Moreover, M2s are involved in the clearance of pathogens and debris, a process essential for initiating tissue repair [52].M2s are central to the process of lung repair, contributing to the resolution of inflammation, clearance of debris, and tissue remodeling.This particular context could potentially offer a favorable outlook when interpreting the rise of macrophages following MAGN treatment as advantageous.SiglecF + Gr1 hi eosinophils also uniquely express lymphocyte-targeting cytokines such as CXCL13 and IL-27, which are involved in the regulation of B and T lymphocyte functions, further indicating their capacity to modulate immune responses in a manner that could exacerbate inflammatory conditions [39].IL-27 targets Tregs for anti-inflammatory functions in allergic inflammation, and altered IL-27 responsiveness in Tregs may perpetuate inflammation [53].The administration of IL-27 via intranasal route has been shown to mitigate Th2-mediated allergic lung inflammation and restructuring in murine asthma models through the restoration of both STAT1 and STAT3 signaling pathways [54].
The elevated production of IL-13 is able to suppress Th17 responses by direct inhibition of IL-23, IL-1beta, and IL-6 expression (according to Kleinschek et al., 2007) [55].Moreover, alternative macrophage activation is induced by type 2 cytokines like IL-13 [48,49].Alternatively activated macrophages secrete anti-inflammatory cytokines that dampen the inflammatory response, thereby promoting epithelial regeneration and alveolar structure remodeling, crucial for the recovery phase of ARDS [50].A recent study by Percopo et al. (2016) showed that the elevated level of Il-13 concentration can be primarily linked to SiglecF + Gr1 hi eosinophils, among various other sources [39].We assume an increased ratio of this distinct subpopulation of eosinophils as explanation for the high levels of IL-13 in BALF of the MAGN-treated group.However, it remains for further research to confirm by flow cytometry the presence of Gr1 antigen (the marker for SiglecF + Gr1 hi eosinophils subpopulation) on the surface of eosinophils and, eventually, the quantification of it by the FACS (fluorescence-activated cell sorting) technique.If there is any causal relation between MAGN and the increased presence of SiglecF + Gr1 hi eosinophils, additional studies must be conducted to ascertain the molecular targets responsible for this presumed phenomenon.
Contrary to expectations, the combination of MAGN pretreatment and SECU was found to be ineffective in reducing IL-6 compared to SECU treatment alone.Moreover, this association decreased SECU potency to diminish IL-6 in BALF.Clinically, the role of IL-6 in ALI is well documented.IL-6's association with the oxygenation index in ALI patients indicates its role as a predictive biomarker for disease severity, where elevated levels correlate with worsened oxygenation, suggesting a direct impact on the physiological severity of lung injury [56].Elevated plasma IL-6 levels were also associated with a decreased number of ventilator-free days (VFDs), highlighting its prognostic value in predicting more severe outcomes and longer dependency on mechanical ventilation in ALI patients [56].The significant association of IL-6 with mortality in a larger cohort within the study underscores its critical role in the pathogenesis of ALI and its potential utility in identifying patients at higher risk of death, thereby guiding more aggressive or targeted therapeutic interventions [56].In rodent models of sepsis [57] and LPS-induced mastitis [58], MAGN exhibited a dose-dependent anti-inflammatory effect.Moreover, increased levels of TNF-α, IL-1β, and IL-6 in lungs were markedly reduced by magnolol in LPS-induced ALI in rats [30].
The elevated IL-4 in LTH induced by MAGN either in monotherapy or combined with SECU might be an important contributor for the shift of macrophages towards the M2 subtype responsible for the production of IL-13, IL-5, TGFβ, and IL-10 [49].Only in LTH do our results demonstrate the ability of combined SECU and MAGN treatment to induce a significant increase of IL-5-the cytokine predominantly responsible for differentiation of eosinophils in the anti-inflammatory CD101 − phenotype [33].
The combination of SECU and MAGN treatments induced IL-13 increase both in BALF and LTH.Besides alternative macrophage activation, elevated production of IL-13 is required for suppression of Th17 responses by direct inhibition of IL-23, IL-1β, and IL-6 expression in activated dendritic cells [55].
In contrast to SECU, MAGN has the potential to boost IFN-γ in both BALF and LTH even in association with SECU.This effect may be beneficial due to the protective role of IFN-γ in cytokine release syndrome-induced extrapulmonary ALI by modulating immune responses and reducing tissue damage [59].
Both studied treatments, either as monotherapies or combined, exhibited an inhibitory effect on IL-17.This validates their therapeutic potential in ALI even from the early stages characterized by elevated levels of interleukin-17A (IL-17A) in the blood and BALF [8,9] and an imbalance between Th17 and Treg cells, favoring a Th17 shift [10].
It is important to highlight the significant difference observed in the study designs, which, in addition to the varying doses of MAGN and LPS given, included the differing durations for MAGN pretreatment, with a particular focus on the inclusion of asthma as a preexisting condition solely within our study.

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Histological modifications for MGN-treated groups are consistent with those of Ni et al., who found minor histopathological changes in lungs from mice with LPSinduced ALI under MAGN treatment, especially in inflammatory cell infiltration [28].

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Finally, it should be taken into account that biologic therapies targeting IL-17 axis administration in atopic patients may require more frequent switching among biologics to control their active disease compared to non-atopic patients [60].

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Study limitations: • The resulting values for cytokines in BALF and LTH represent only a status quo at the sampling moment.This restrains our view to a "moment picture" without any information regarding the dynamic evolution; • Results from murine models can present challenges in translation of findings to the intricate and distinct human disease of asthma.Additionally, utilizing exclusively female animals may prompt inquiries regarding the potential impact on male mice; • MGN has downsides like reduced bioavailability in rodents-less than 10% for oral administration [61]-or the potential inhibition of UDP-glucuronosyltransferase (UGT) enzyme activity isoforms 1A7 and 1A9 in humans and rodents [62,63].Inhibition of UGTs by magnolol may be a potential mechanism that enhances the toxicity of drugs or other active compounds contained in the herbal preparation.Because of these disadvantages, future research should consider natural sources for drugs with a higher variability and abundance, like marine drugs [64].

Conclusions
The combined treatment with SECU and MAGN in ALI models shows a complex immunomodulatory impact, with both beneficial and detrimental effects.It promotes M2 macrophage polarization, enhancing production of anti-inflammatory cytokines such as IL-4, IL-5, IL-10, and IL-13, which are crucial for lung repair and inflammation resolution.Additionally, it inhibits IL-17, reducing early-stage inflammation.MAGN pretreatment alone demonstrates a higher potency in reducing neutrophils and enhancing IFN-γ, suggesting its potential in mitigating severe asthma symptoms and modulating immune responses.On the other hand, while both treatments inhibit IL-17 and promote M2 macrophage polarization, the combination may exacerbate allergic responses and increase OVA-specific IgE, potentially worsening ALI outcomes.Moreover, the combination treatment's inability to reduce IL-6 and its potential to exacerbate allergic inflammation highlight the need for careful consideration in therapeutic applications.These findings could offer valuable insights for the advancement of precision medicine within the realm of respiratory illnesses.

Figure 3 21 Figure 3 .
Figure 3 reveals increased OVA-specific IgE concentration in all OVA-exposed groups, confirming the OVA sensitization.The apparent benefits of MAGN cannot be confirmed due to the lack of statistical significance from the OVA + LPS (positive disease control group) or secukinumab-treated group.Surprisingly, the association of MAGN and SECU treatments seems to determine an increase of OVA-specific IgE serum concentration in comparison to all other OVA-exposed groups.Biomedicines 2024, 12, x FOR PEER REVIEW 7 of 21

Biomedicines 2024 , 21 Figure 8 .
Figure 8. Representative HE-and PAS-stained tissue sections of lungs and bronchia from mice in ovalbumin-induced asthma exacerbated with LPS administration.Original magnification for HE: ×40 or ×100, for PAS: ×1000.SAL: normal control group; OVA + LPS: positive disease control group; OVA + LPS + MAGN: magnolol-treated group; OVA + LPS + SECU: secukinumab-treated group; OVA + LPS + MAGN + SECU: combined magnolol-and secukinumab-treated group.In the context of SECU treatment only, a notable reduction is observed in the quantity of neutrophils, persisting at diminished levels in the vicinity of the bronchi.The pulmonary alveoli exhibit a diminished thickness in their walls and a decreased presence of figurative elements within the lumen or septal structures, and instances of alveolar collapse are infrequent.
" University of Medicine and Pharmacy of Iaşi, Romania approved the study design and protocols (approval No.